Leukemia is a disease caused by canceration of hematopoietic stem cells in bone marrow. In particular, chronic myeloid leukemia (CML) is known to be caused by the bcr-ab1 fusion gene formed by translocation between chromosome 9 and chromosome 22, and an ABL kinase inhibitor imatinib or the like is widely used for its therapy. However, there is a problem in that, in cases where a point mutation exists in this ab1 gene (including the ab1 gene in the fusion gene), resistance to imatinib is expressed. In such cases, the dose of imatinib needs to be increased; the therapeutic agent needs to be changed; or the therapy needs to be switched to bone marrow transplantation. Therefore, in therapy of leukemia, especially CML, it is very important to detect the presence/absence of a point mutation in the ab1 gene.
Examples of the method for detecting a point mutation in the ab1 gene include a method wherein an ab1 gene mutation is amplified by RT-PCR and cloned, followed by sequencing analysis thereof (PNAS Aug. 6, 2002 vol. 99 no. 16 10700-10705); a method wherein an ab1 gene mutation is detected by the PCR-RFLP method (Leukemia (2002) 16, 2190-2196), and a method wherein an ab1 gene mutation is detected by the DHPLC method and subjected to sequencing analysis (Clin Cancer Res 2006; 12(24) Dec. 15, 2006). However, the method described in PNAS 2002 requires expertise and skill for the experimental operation, and automation is difficult. Further, the experimental operation is laborious, and it takes several days to obtain a result. The method described in Leukemia 2002 also requires laborious operation, and it takes about 1 day to obtain a result. Further, the amplification product may cause contamination in another reaction. Further, its automation is difficult. The method described in Clin Cancer Res 2006 also requires laborious operation, and its automation is difficult. Further, in the DHPLC method, discrimination of mutant types is difficult, so that the mutant type needs to be separately detected by sequencing or the like.
Because of these problems, in recent years, as a method of detecting a polymorphism, detection using melting curve analysis (Tm analysis) has been carried out (JP 2001-286300 A, JP 2002-119291 A). In this method, a probe complementary to the sequence to be detected which contains a genetic polymorphism to be detected is used and a target single-stranded DNA in the detection sample is allowed to form a hybrid (double-stranded DNA) with the probe. This hybrid-forming body is subjected to heat treatment, and dissociation (melting) of the hybrid due to increased temperature is detected by measurement of a signal such as the absorbance. Based on the detection result, the Tm value is determined, thereby judging the presence/absence of the polymorphism of interest. The Tm value increases as the homology in the hybrid-forming body increases, while the value decreases as the homology decreases. Therefore, the Tm value (evaluation criterion value) of the hybrid-forming, body between the sequence to be detected containing the polymorphism and of interest and the probe complementary thereto is preliminarily determined, and the Tm value (measure value) between the target single-stranded DNA in the detection sample and the probe is measured, and, in cases where the measured value is the same as the evaluation criterion value, the result can be judged as “matching”, which means that the target DNA has the polymorphism of interest, while in cases where the measured value is lower than the evaluation criterion value, the result can be judged as “mismatching”, which means that the target DNA does not have the polymorphism of interest.
However, there is a problem in that such a detection method using Tm analysis has a low sensitivity. This is especially problematic when a point mutation is to be detected from DNA derived from blood cells of a leukemia patient (Japanese Translated PCT Patent Application Laid-open No. 2004-537992). That is, even in blood of a single CML patient, blood cells having a point mutation generated in the ab1 gene (mutant gene) and blood cells having no such mutation (normal gene) are contained, and the only difference between them is a point mutation, that is, the sequence of a single nucleotide. This leads to a phenomenon wherein the probe for detection of a point mutation hybridizes (matches) with a mutant sequence having the point mutation (sequence to be detected), while hybridizing (mismatches) also with the normal sequence which does not have the point mutation (sequence not to be detected). In such cases, there is a problem in that, when the melting curve indicating the relationship between the signal strength and the temperature is prepared by the Tm analysis, detection of the peak in the higher-temperature side, which is the peak for the matched mutant sequence, is difficult because of the existence of the peak in the lower-temperature side, which is the peak for the mismatched normal sequence. That is, in conventional probes, there is a problem in that, even in cases where a mutant sequence containing a mutation exists, its detection is difficult because of the existence of the normal sequence which does not contain the mutation, so that the detection sensitivity decreases.
In WO2008/018305, probes to be employed in a detection method using Tm analysis are described, and those probes are used to detect A758T(Y253F), G756C(Q250E), G763A(E255K) and A758T(Y253F), which are point mutations in the ab1 gene. Further, in JP 2008-199965 A, probes for detecting A730G(M244V), G749A(G250E), A943 G(T315A), C944T(T315I), C951G(F317L), T1052C(M351T). A1064G(E355G), T1075G(F359V) and A1187G(H396R) are described. However, probes for detecting T10760 (F359C), T757C (Y253H), A764T (E255V) and G895C/T (V299L) have not been described in any literature.